Role of biokinetics in risk assessment of drugs and chemicals in children.

Whether children incur different risks from xenobiotics than adults will depend on the exposure, biokinetics, and dynamics of compound. In this paper, current knowledge on developmental physiology and possible effects on biokinetics are evaluated and the role of biokinetics in risk assessment both for drugs and chemicals is discussed. It is concluded that most dramatic age-related physiological changes that may affect biokinetics occur in the first 6-12 months of age. The difference in internal exposure between children and adults can generally be predicted from already known developmental physiological differences. However, for risk assessment it will also be necessary to determine whether internal exposure is within the drug's therapeutic window or if it will exceed the NOAEL of a chemical. Furthermore, the effects of internal exposure of potentially harmful compounds on developing organ systems is of utmost importance. However, knowledge on this aspect is very limited. Risk assessment in children could be improved by: (1) application of pediatric PBPK-models in order to gain insight into internal exposure in children, (2) studies in juvenile animals for studying effects on developing systems, and (3) extrapolation of knowledge on the relationship between internal exposure and dynamics for drugs to other chemicals.

Transport and metabolism of glutathione conjugates of menadione and ethacrynic acid in confluent monolayers of rat renal proximal tubular cells.

Confluent monolayers of primary rat renal proximal tubular (RPT) cells were used to compare transepithelial transport and concomitant metabolism of two different glutathione (GSH) S-conjugates. For the GSH-conjugated quinone compound, [35S]GSH-conjugated menadione (MGNQ), no specific transepithelial transport was observed. Most likely, [35S]MGNQ passed the monolayer via paracellular leakage as the result of a reduction in monolayer integrity due to toxicity via extensive redox cycling of the quinone under the culture conditions. RPT cell monolayers metabolise MGNQ into a cysteinylglycine conjugate, which after intramolecular cyclization yields 2H-(3-glycinyl)-9-hydroxy-10-methyl-1,4-naphthothiazine. Acivicin, an inhibitor of gamma-glutamyltranspeptidase, inhibited the formation of this 1,4-napthothiazine adduct. The second product formed is 1,4-napthothiazine formed by loss of glycine via the action of dipeptidases. Similarly, no basolateral (B) to apical (A) transport of a GSH-conjugated alpha, beta unsaturated ketone, [14C]ethacrynic acid (EASG), occurred. However, net transport of [14C] radioactivity could be observed from A=>B direction. After 8 h, 23% of total [14C] radioactivity was transported from the apical to the basolateral chamber. In both the apical and basolateral chambers, free, unconjugated ethacrynic acid (EA) was observed. gamma GT-mediated metabolism of EASG to the much more unstable cysteinylglycine conjugate leads to relatively large amounts of free EA. Thus, the GSH conjugate is not transported but rather the cysteine adduct and/or free, unconjugated EA. In agreement with this, acivicin reduced A=>B transport of EASG and inhibited the formation of free EA. In conclusion, the confluent monolayers of RPT cells do not or no longer possess active basolateral transport systems for GSH conjugates. However, they are still quite useful for studying biotransformation reactions of thioether conjugates.

Cytotoxicity of 2-tert-butyl hydroquinone glutathione conjugates after apical and basolateral exposure of rat renal proximal tubular cell monolayers.

Confluent monolayers of renal proximal tubular (RPT) cells cultured on porous supports were used to investigate the cytotoxicity induced by glutathione conjugates of 2-tert-butyl-(1,4)-hydroquinone (SG-TBHQ) after apical and basolateral exposure. As judged from lactate dehydrogenase (LDH) leakage, cytotoxicity was observed after basolateral exposure of monolayers to 250 and 500 mum 2-tert-butyl-5-(glutathion-S-yl)hydroquinone (5SG-TBHQ). In these experiments, LDH leakage was 22.3 +/- 1.9 and 32.2 +/- 1.9%, respectively. Basolateral exposure of monolayers to 250 and 500 mum 6SG-TBHQ resulted in LDH leakage of 22.2 +/- 2.5 and 30.0 +/- 2.7%, respectively. The double conjugate, 2-tert-butyl-3,6-(diglutathione-S-yl)hydroquinone (3,6SG-TBHQ), was not toxic and LDH leakage was about control level (15.0%). Basolaterally located probenecid-sensitive organic anion transporters did not seem to play a part in the cytotoxic effect. However, when RPT cell monolayers were cultured in 24-well tissue culture plates, apical challenge with 250 mum 5SG-TBHQ induced a cytotoxic effect. In these experiments, LDH leakage was 33.5 +/- 0.6%. With these cells, inhibition of apical gamma-glutamyltranspeptidase (gammaGT) activity by acivicin, which was not toxic by itself, decreased 5SG-TBHQ-induced LDH leakage to 19.3 +/- 1.2%, whereas 6SG-TBHQ (also 250mum)-induced LDH leakage was increased to 55.3 +/- 1.0%. Co-incubation of RPT cells with SG-TBHQs in the presence of 1.5 mm ascorbic acid (AA) pointed to a pro-oxidant rather than an antioxidant effect of AA. Superoxide dismutase and catalase completely abolished SG-TBHQ-induced cytotoxicity. Since cultured RPT cells lack N-acetylation of cysteine conjugates, the N-deacetylation/N-acetylation ratio cannot have a vital role in renal toxicity of quinone thioethers in this in vitro system. It seems, therefore, that the cytotoxicity observed is mainly the result of extracellular redox cycling of SG-TBHQs. The lack of toxicity of 6SG-TBHQ after apical exposure could be due to detoxification by gammaGT-mediated cysteinylglycine- or cysteine-conjugate formation followed by cyclization, as shown for other related quinone glutathione conjugates. The relative importance of the observed effects for the in vivo situation is discussed.

In vitro metabolism of 5-fluoro-2-glutathionyl-nitrobenzene by kidney proximal tubular cells studied by 19F-NMR.

Proximal tubular biotransformation of the glutathionyl (GSH) conjugate derived from 2,5-difluoronitrobenzene (5-fluoro-2-glutathionyl-nitrobenzene) was studied by means of 19F-NMR. This method allows a direct and specific detection of the fluorinated metabolites formed, at a detection limit of 1 microM for an overnight NMR run. Incubation of a monolayer of LLCPK1 cells with 100 microM 5-fluoro-2-glutathionyl-nitrobenzene for 24 h showed that these cells metabolize this GSH conjugate into the corresponding cysteinylglycyl and cysteine conjugate. The expected N-acetylcysteine conjugate however was not formed as an endproduct. Additional experiments demonstrated the absence of N-acetyltransferase activity in LLCPK1 cell lysates incubated with FCysNB and also the rapid loss of this activity in isolated renal proximal tubular cells (RPT): freshly isolated RPT cells do convert FCysNB to FNAcNB as major metabolite but, upon cultivation, quickly lose this capacity. Since uptake of FCysNB might also be a limiting factor, we investigated transport of FCysNB from the apical to the basolateral side of the culture RPT cells. No indication for such transport was obtained. Thus, the absence of mercapturic acid formation in LLCPK1 cells and cultured RPT cells is the results of a decline in N-acetyltransferase activity and perhaps a deficient cellular uptake of the cysteine conjugate.

Regulation of interrenal function in freshwater and sea water adapted tilapia (Oreochromis mossambicus).

In teleosts, cortisol is one of the key factors regulating the adaptation to environmental challenges, such as salinity changes. This paper compares interrenal function between fully adapted freshwater (FW) and sea water (SW) specimens of the euryhaline teleost Oreochromis mossambicus (tilapia), combining morphometric and biosynthetic approaches. Interrenal tissue and two tissues producing interrenal secretagogues (ACTH and ANP; atrial natriuretic peptide) were studied. The results demonstrate that sea water adaptation concurs with a sustained stimulation of the interrenal cells, as evidenced by a marked hyperplasia of the cells and the higher initial ex vivo cortisol release in seawater adapted tilapia. This difference was not reflected in ultrastructural differences in the pituitary corticotropes. Plasma ACTH levels were also similar in FW and SW adapted tilapia. Moreover, in vitro data indicate that the ACTH sensitivity of the interrenal cells of both groups was also similar. A second potential interrenal secretagogue (ANP) has recently been implicated in teleost ionic regulation during salinity changes. However, plasma immunoreactive ANP levels and in vitro production of the hormone were also indistinguishable between FW and SW tilapia. ANP pretreatment of tilapia head kidneys in vitro strongly inhibited the response to ACTH, an effect previously undocumented for teleosts. Whereas the sustained stimulation of the interrenal cells under sea water conditions corroborates results obtained with other teleost species, thereby supporting a hypoosmoregulatory role for cortisol, it also is evident that notable species differences exist regarding the regulation of the interrenal gland under these conditions.

Differential detoxification of two thioether conjugates of menadione in confluent monolayers of rat renal proximal tubular cells.

This in vitro study describes proximal tubular toxicity of quinone thioethers after incubation of these compounds on either side of a renal proximal tubular cell (RPTC) monolayer. These cells were cultured to confluency on porous supports of tissue culture inserts, and the apically and basolaterally induced toxicity of two thioether conjugates of menadione (2-methyl-1,4-naphthoquinone) was investigated. As judged by lactate dehydrogenase (LDH) leakage, the glutathione (GSH) conjugate of menadione (MGNQ) was only toxic after basolateral challenge. However, after inhibition of gamma-glutamyl transpeptidase by acivicin, MGNQ was also toxic after apical challenge. The mercapturic acid of menadione [M(NAC)NQ] displayed cytotoxicity both after apical and basolateral challenge. From the basolateral side, MGNQ and M(NAC)NQ-induced cytotoxicity could be enhanced by inhibition of the organic anion transport system with probenecid. Inhibition of beta-lyase did not influence M(NAC)NQ-induced cytotoxicity. In addition, inhibition of intracellular N-deacetylation of M(NAC)NQ using paraoxon potentiated the observed toxic effect. Thus, it appears that the MGNQ and M(NAC)NQ-induced cytotoxicity is the result of extracellular events, presumably redox cycling. Putative uptake of the conjugates is likely to be associated with detoxification of these compounds.

Regulation of differential release of alpha-melanocyte-stimulating hormone forms from the pituitary of a teleost fish, Oreochromis mossambicus.

Using high-performance liquid chromatography (HPLC) in combination with radioimmunoassay, three forms of alpha-MSH (des-acetyl, mono-acetyl and di-acetyl alpha-MSH) were separated and identified in tilapia neurointermediate lobes and plasma, and in medium from lobes superfused in vitro. The presence of acetylated forms in lobe extracts indicated that the peptides are acetylated intracellularly. Di-acetyl alpha-MSH was, especially in comparison with monoacetyl alpha-MSH, relatively more abundant in lobe extracts than in plasma. This suggests that the three forms of alpha-MSH are not released according to their relative intracellular abundances. The possibility of regulation of this differential release by dopamine and TRH was investigated, using a microsuperfusion system. Dopamine was a potent inhibitor of alpha-MSH release, but did not modulate the relative abundance of the different forms of alpha-MSH released from the MSH cells. TRH was a potent stimulator of alpha-MSH release. It enhanced in vitro the release of di-acetyl alpha-MSH more than the release of mono-acetyl alpha-MSH. Thus tilapia may be able to modulate not only the quantitative but also the qualitative signal from the MSH cells. This might enhance the flexibility of the animals to respond to environmental challenges.

Phorbol ester inhibits cholecystokinin octapeptide-induced amylase secretion and calcium mobilization, but is without effect on secretagogue-induced hydrolysis of phosphatidylinositol 4,5-bisphosphate in rabbit pancreatic acini.

The effect of prolonged protein kinase C activation on cholecystokinin octapeptide (CCK-8)-induced amylase secretion from rabbit pancreatic acini was studied by means of the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA). The phorbol ester itself increased basal amylase secretion but inhibited completely the secretory response to relatively low concentrations of CCK-8. The inhibitory action of TPA on CCK-8-induced amylase secretion was paralleled by inhibition of CCK-8-induced calcium mobilization but not by inhibition of CCK-8-induced breakdown of 32P-labelled phosphatidylinositol 4,5-bisphosphate. The results presented suggest that protein kinase C, or one of its phosphorylated products, inhibits the CCK-8-stimulated pathway leading to secretion at a level beyond the secretagogue-induced hydrolysis of phosphatidylinositol 4,5-bisphosphate. Inhibition of the initial, inositol 1,4,5-trisphosphate-mediated and extracellular calcium-independent, increase in free cytosolic calcium concentration, together with the findings of others, suggests that the efficacy of this inositol-phosphate to release calcium is reduced.